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  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
  • C07K16/3084—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated gangliosides
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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
  • C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• the present invention relates to an anti-galectin-9 antibody and an inhibitor of regulatory T-cell suppressive activity, and to the use of this antibody for the treatment of diseases associated with the suppressive activity of regulatory T-cells.
• Human T lymphocytes are characterized by the expression of a membrane marker called CD3 and a specific receptor, the TCR (T cell receptor), which is directly involved in the specific recognition of an antigen.
• TCR T cell receptor
• CD4 + T lymphocytes also called helper T cells, secrete major cytokines that help in particular B cells in their humoral function (production of specific antibodies) and CD8 + T lymphocytes in their cytotoxic activity.
• CD4 + T cells Another population of CD4 + T cells consists of natural regulatory T cells, hereafter referred to more briefly as “regulatory T cells”. They overexpress constitutively the CD25 molecule (the regulatory T lymphocytes can thus also be called “CD4 + CD25 +”) and the Foxp3 transcription factor. This low percentage of CD4 + CD25 + T cells has the particularity of negatively regulating the actors of the immune response who would have recognized various autoantigens by their TCRs. Regulatory T lymphocytes thus play a major role in the physiology of the immune system, in particular to protect the body against the emergence of autoimmune diseases.
• regulatory T lymphocytes can induce inappropriate immunosuppression, which then promotes tumor growth or the persistence of infectious pathogens (viruses, bacteria, parasites, etc.). Numerous studies have thus shown that regulatory T cells decrease anti-tumor or anti-viral immune responses, in particular by inappropriately inhibiting the activity of effector T cells, thus favoring the persistence of viruses and the tumor progression in a large scale. majority of cancers.
• regulatory T cells exert their suppressive effects on effector T cells
• various studies have highlighted different mechanisms by which regulatory T cells could suppress the immune response.
• Foxp3 + regulatory T lymphocytes can lyse effector T lymphocytes via the production of granzymes / perforins (1) or by labeling effector lymphocytes to IL-2 or by inhibiting the proliferation of effector T cells, in particular by expressing surface molecules such as galectin 1 which interacts with receptors expressed on effector T lymphocytes and induces cell cycle arrest of effector T lymphocytes (2).
• Treg The pathophysiological role of Treg in cancers has thus encouraged the emergence of a new anti-tumor therapeutic strategy. It consists in neutralizing the inhibitory factors of the immune response, and in particular the regulatory T lymphocytes, or in other words to break the tolerance towards the tumor antigens.
• rodent analyzes have shown that inhibition of CD4 + CD25 + regulatory T cells by a monoclonal antibody directed against the IL-2 alpha receptor (CD25) promotes activation and expansion of T cells. effector inhibiting tumor growth (3).
• CD25 is also expressed by activated effector T cells. So this strategy is to be taken with caution in the as it can also promote the elimination of effector T cells.
• GITR signaling via an anti-GITR antibody is capable of inhibiting the suppressive activity of regulatory T cells (4).
• use of an anti-GITR antibody in the treatment of murine tumors made it possible to increase the anti-tumor response of the CD4 + and CD8 + T lymphocytes, and this more effectively when the tumor is already installed.
• activated effector T cells also express GITR. Therefore, there is also a risk of effector T-cell suppression when using an anti-GITR antibody.
• the strategy to inhibit regulatory T cells has also been contemplated by the use of an anti-CTLA4 antibody, a marker expressed by regulatory T cells.
• an anti-CTLA4 antibody a marker expressed by regulatory T cells.
• CTLA-4 KO KnockOut
• an increase in lymphocyte activity was shown T effectors and a decrease in suppression mediated by regulatory T cells, leading to inhibition of tumor growth (5).
• activated effector T cells also express the CTLA-4 marker. Therefore, again, a risk of suppression of effector T cells by the use of an anti-GITR antibody.
• regulatory T cells are also markers of activation of effector T lymphocytes.
• the use of these proteins as targets for the depletion of regulatory T cells thus has the undesirable effect of eliminating numerous CD4 + and CD8 + effector T cells, which are essential for tumor regression. In this context, it remains very difficult to specifically target regulatory T cells in therapeutic protocols.
• One of the aims of the invention is thus to provide an antibody which is directed against a marker specific for regulatory T cells and which allows the inhibition of the suppressive activity of regulatory T cells, without altering the function of effector T cells.
• the inventors have the merit of having demonstrated that a molecule, galectin 9, is expressed specifically by regulatory T cells during activation and that an antibody directed against this molecule makes it possible to inhibit the suppressive activity.
• regulatory T lymphocytes and this specifically, that is to say without inhibiting effector T cells.
• such an antibody is capable of neutralizing the conversion of conventional CD4 + T lymphocytes into immunosuppressive CD4 + T induced by galectin 9, thus favoring the maintenance of an anti-tumor immune response in the treated patient. according to the invention.
• the term "disease associated with the suppressive activity of regulatory T cells” is intended to mean any (non-autoimmune) disease in which the suppressive activity of regulatory T lymphocytes plays a role, in particular by promoting the development or the persistence of the disease. .
• the suppressive activity of regulatory T cells has been shown to promote the development of tumors. The invention therefore more particularly targets cancers in which the suppressive activity of T lymphocytes plays a role.
• Galectins, or type S lectins constitute a family consisting of fifteen members in vertebrates, including ten in humans.
• Galectin 9 interacts preferentially with the beta-galactoside residues of glycoproteins and glycolipids. In humans, galectin 9 exists in three isoforms, long, medium and short.
• Several studies have been conducted to determine the link between the development of cancers and galectins, including galectin 9. However, it is considered in most of these studies that galectin 9 has a cytotoxic activity against activated T cells ( whether CD4 + or CD8 +) and has no cytotoxic activity against unactivated T cells.
• patent application EP1586325 is based on the assumption that, in vitro, galectin 9 induces the apoptosis of tumor cells, in particular in malignant or metastatic cells, but not normal cells.
• the application EP1586325 thus targets a drug comprising galectin 9 or molecules inducing the production and / or release of galectin 9, etc.
• the objective was therefore to use galectin 9 or factors making it possible to increase galectin 9, while the present invention tends, on the contrary, to inhibit it.
• the present invention thus relates to an antibody directed against galectin 9 and an inhibitor of the suppressive activity of regulatory T cells.
• the invention relates to an antibody directed against galectin 9, characterized in that it is an inhibitor of the suppressive activity of regulatory T cells.
• the invention is based on the unexpected findings made by the inventors, who have observed (i) on the one hand, that galectin 9 is directly expressed by regulatory T cells and that its expression is increased during their activation and (ii) on the other hand, that galectin 9 is very weakly expressed by effector T cells and that this expression disappears during activation. Moreover, the inventors have observed that the inhibition of galectin 9 by an antibody allows the inhibition of the suppressive activity of regulatory T cells.
• regulatory T cells is meant the subregion of naturally occurring regulatory T cells, also called Treg, characterized by constitutive expression of CD25, CTLA-4, and GITR and by specific expression of the Foxp3 transcription factor. Regulatory T lymphocytes more particularly targeted by the present invention so are the natural regulatory T cells, or nTreg.
• the term "suppressive activity of regulatory T cells” is understood to mean the immunosuppressive activity that regulatory T cells exert on effector T lymphocytes, once activated, in a pathological situation and which particularly favors tumor growth.
• the suppressive activity of regulatory T cells can be understood as the activity decreasing anti-tumor immune responses by inhibiting the activity of effector T cells.
• the suppressive activity of regulatory T cells can be analyzed according to various techniques known to those skilled in the art. For example, a MLR (Mixed Leukocyte Reaction) method may be performed, which implements a co-culture of autologous or heterologous Treg lymphocytes and immune cells (total PBMC or CD4 + T).
• radioelements such as Tritiated Thymidine
• EdU 5-ethynyl-2'-deoxyuridine
• the inhibitory effect of an antibody according to the invention can thus for example be analyzed by such an MLR method, carried out in the presence of the antibody tested.
• antibodies and “immunoglobulin” are used interchangeably and refer to immunoglobulin molecules or immunologically active portions of immunoglobulin molecules, i.e. molecules comprising the specific binding sites of a given antigen.
• the term antibody covers not only whole antibody molecules but also antibody fragments and variants (including derivatives such as humanized antibodies) of antibodies and antibody fragments.
• Immunoglobulins are well known to those skilled in the art and consist of two heavy chains connected to each other by disulfide bridges, each heavy chain being connected to a light chain by a disulfide bridge.
• Each string contains separate sequence domains.
• the light chain comprises two domains, a variable domain (or region) (VL) and a constant domain (CL).
• the heavy chain comprises four or five domains according to the classes of antibodies, a variable domain (VH) and three or even four constant domains (CH1, CH2, CH3 and optionally CH4).
• VH variable domain
• CH1, CH2, CH3 and optionally CH4 constant domains
• the constant domains of the light (CL) and heavy (CH) chains confer on the antibody important biological properties such as the association of the antibody chains with each other, the mobility through the placenta, the fixation of the complement and / or the attachment to the antibodies.
• Fc receptors FcR
• the Fv fragment corresponds to the V-terminal part of the Fab fragment, described below, of the immunoglobulin, and comprises the variable portions of a light chain and a heavy chain (VL and VH).
• the specificity of the antibody lies in the structural complementarity between the recognition site of the antibody and the antigenic determinant.
• the antibody recognition site consists essentially of residues from hypervariable or complementarity determining regions (CDRs).
• CDRs complementarity regions
• FR framework regions
• An antibody according to the invention may be a monoclonal or polyclonal antibody.
• an antibody according to the invention is a monoclonal antibody.
• the term "monoclonal antibody” or “mAb” refers to an antibody of unique amino acid composition, which is directed against a specific antigen and can be produced by a single B cell clone, or hybridoma.
• Monoclonal antibodies can also be recombinant, i.e., be produced by protein engineering techniques.
• Fab refers to an antibody fragment of about 50,000 dalton molecular weight and having antigen binding activity. It comprises about half of the N-terminal side of the heavy chain and the entire light chain bound by a disulfide bridge.
• the Fab can be obtained in particular by the immunoglobulin treatment with a protease, papain.
• F (ab ') 2 refers to a fragment of about 100,000 dalton and antigen binding activity. This fragment is slightly larger than two Fab fragments connected via a disulfide bridge in the hinge region. These fragments are obtained by treatment of an immunoglobulin with a protease, pepsin. The Fab fragment can be obtained from the F (ab ') 2 fragment by cleaving the disulfide bridge of the hinge region.
• a single Fv chain "scFv” corresponds to a VH: VL polypeptide synthesized using the genes encoding the VL and VH domains and a sequence encoding a peptide for binding these domains.
• An scFv according to the invention includes CDRs maintained in a suitable conformation, for example using genetic recombination techniques.
• the dimers of "ScFv” correspond to two molecules of scFv linked together by a peptide bond. This Fv chain is frequently the result of the expression of a fusion gene including the genes encoding VH and VL linked by a linker sequence encoding a peptide.
• the human scFv fragment may include CDRs regions that are maintained in an appropriate conformation, preferably through the use of genetic recombination techniques.
• the "dsFv” fragment is a VH-VL heterodimer stabilized by a disulfide bridge; it can be divalent (dsFV2).
• Sc (Fv) 2 or multivalent divalent antibody fragments may be formed spontaneously by association of monovalent scFvs or produced by linking scFvs fragments by binding sequences peptide.
• the Fc fragment supports the biological properties of the antibody, in particular its ability to be recognized by immunity effectors or to activate complement. It consists of the constant fragments of the heavy chains beyond the hinge region.
• diabodies means small antibody fragments having two antigen binding sites. These fragments comprise in the same VH-VL polypeptide chain a VH heavy chain variable domain connected to a VL light chain variable domain. By using a binding sequence that is too short to allow pairing of two domains of the same chain, pairing with two complementary domains of another chain necessarily occurs and thus two antigen binding sites are created.
• An antibody of the invention may thus be an immunoglobulin consisting of two heavy chains and two complete light chains, or may be an immunoglobulin fragment according to the invention, for example F (ab ') 2, Fab, Fv, scFv or Fc.
• an antibody fragment is the Fab region of an immunoglobulin, in particular the Fv region of an IgG1 antibody.
• the antibodies described in the invention are isolated and purified, and are different from natural antibodies.
• the terms “isolated” and “purified” indicate that the molecule is present in the major absence of other biological macromolecules of the same type.
• chimeric antibody refers to an antibody in which the sequence of each light chain and / or heavy chain constituting it comprises or consists of a hybrid sequence derived from at least two distinct animals.
• the chimeric antibodies of the invention are human / mouse hybrids.
• a chimeric antibody of the invention may comprise a VH domain and a VL domain of an antibody originating from a non-human animal, in particular a murine animal, and a CH domain and a CL domain of a human antibody.
• an antibody of the invention comprises a VH domain and a VL domain of an antibody derived from the 1G3 antibody, defined below, and a CH domain and a CL domain of a human antibody.
• the term “humanized antibody” refers to an antibody derived from a non-human animal in which heavy and light chain sequences other than CDRs have been replaced by corresponding sequences of one or more antibodies of human origin.
• the term “humanized antibody” refers to an antibody whose heavy chain and light chain sequences are of human origin, and whose CDRs are derived from the 1G3 antibody.
• the antibodies according to the invention are preferably monoclonal antibodies, that is to say that they recognize only one antigenic determinant in galectin 9, unlike polyclonal antibodies which correspond to a mixture of monoclonal antibodies, which therefore recognize several antigenic determinants in the same molecule.
• Monoclonal antibodies according to the invention can be obtained according to the techniques well known to those skilled in the art. For example, it is possible to use the cell fusion technique, the heavy and light chain sequence cloning technique, the display phage or ribosome technique, the immunization of mice with the human immunoglobulin repertoire and expression in a ad hoc cell or a transgenic animal. These techniques are well known to those skilled in the art.
• the present invention relates to antibodies directed against galectin 9 and inhibitors of regulatory T cell suppressive activity.
• the inventors have developed a hybridoma producing a murine Iggl Kappa antibody, 1G3, directed against galectin 9 and an inhibitor of the suppressive activity of regulatory T cells.
• the inventors have characterized the variable domains of the light and heavy chains of this monoclonal antibody mAb 1G3 and thus determined the CDRs of this antibody, presented in Table 1.
• H-CDR3 HGGYDGFDY (SEQ ID NO: 4)
• a particular embodiment of the invention therefore relates to an antibody directed against galectin 9 and an inhibitor of the regulatory T cell suppressive activity, having the same binding zone as the 1G3 antibody having for CDRs the six CDRs defined by:
• amino acid sequence SEQ ID NO: 2 in the H-CDR1 region amino acid sequence SEQ ID NO: 2 in the H-CDR1 region
• amino acid sequence SEQ ID NO: 3 in the H-CDR2 region amino acid sequence SEQ ID NO: 3 in the H-CDR2 region
• amino acid sequence SEQ ID NO: 4 in the H-CDR3 region amino acid sequence SEQ ID NO: 4 in the H-CDR3 region
• amino acid sequence SEQ ID NO: 6 in the L-CDR1 region amino acid sequence SEQ ID NO: 6 in the L-CDR1 region
• amino acid sequence SEQ ID NO: 7 in the L-CDR2 region is the amino acid sequence SEQ ID NO: 7 in the L-CDR2 region
• amino acid sequence SEQ ID NO: 8 in the L-CDR3 region is the amino acid sequence SEQ ID NO: 8 in the L-CDR3 region.
• an object of the invention relates to an antibody directed against galectin 9 and inhibitor of the suppressive activity of regulatory T lymphocytes having for CDRs the six CDRS defined by:
• amino acid sequence SEQ ID NO: 2 in the H-CDR1 region amino acid sequence SEQ ID NO: 2 in the H-CDR1 region
• amino acid sequence SEQ ID NO: 3 in the H-CDR2 region the amino acid sequence SEQ ID NO: 4 in the H-CDR3 region, the amino acid sequence SEQ ID NO: 6 in the L-CDR1 region,
• amino acid sequence SEQ ID NO: 7 in the L-CDR2 region is the amino acid sequence SEQ ID NO: 7 in the L-CDR2 region
• amino acid sequence SEQ ID NO: 8 in the L-CDR3 region is the amino acid sequence SEQ ID NO: 8 in the L-CDR3 region.
• the heavy chain variable region of said antibody has the amino acid sequence SEQ ID NO: 1 and the light chain variable region of said antibody has the amino acid sequence SEQ ID NO: 5.
• An antibody according to the invention therefore binds specifically to galectin 9 and is an inhibitor of the suppressive activity of regulatory T cells.
• an antibody according to the invention can bind specifically to an epitope of galectin 9.
• an antibody according to the invention is capable of binding in a specific manner. at the membrane or intracellular galectin 9
• an antibody according to the invention can bind to the epitope recognized by the 1G3 antibody, defined above.
• an antibody according to the invention can bind specifically to the epitope of amino acid sequence SEQ ID NO: 9, presented in Table 2.
• This epitope consisting of the amino acid SEQ ID NO: 9 corresponds to the peptide P4 and covers the end of the binding peptide and the beginning of the C-terminal portion of galectin 9.
• This sequence exists in the three isoforms of galectin 9 (amino acids 166 to 178 of isoform S, amino acids 178 to 190 of isoform M, amino acids 210 to 222 of isoform L).
• Such an antibody can therefore react with all the isoforms of galectin 9.
• the antibody according to the invention is a chimeric antibody, preferably a murine / human chimeric antibody.
• this murine / human chimeric antibody may comprise the variable domains of the 1G3 antibody as defined above.
• the antibody according to the invention is a humanized antibody.
• the variable domain of this humanized antibody may comprise framework regions of the human acceptor, and possibly human constant domains, and the CDRs of the non-human donor, in particular the CDRs defined above.
• the inventors have also developed a hybridoma producing a murine IGgl Kappa antibody, 2E12, directed against galectin 9 and an inhibitor of regulatory T cell suppressive activity.
• the inventors have characterized the variable domains of the light and heavy chains of this monoclonal antibody mAb 2E12 and thus determined the CDRs of this antibody, presented in Table 3.
• H-CDR3 HGGYDGFDY (SEQ ID NO: 13)
• L-CDR1 KS S QSLLYS NNQKN YLA (SEQ ID NO: 15)
• L-CDR2 WASTRGS (SEQ ID NO: 16)
• a particular embodiment of the invention therefore relates to an antibody directed against galectin 9 and an inhibitor of the regulatory T cell suppressive activity, having the same binding zone as the 2E12 antibody having for CDRs the six CDRs defined by:
• amino acid sequence SEQ ID NO: 12 in the H-CDR2 region is the amino acid sequence SEQ ID NO: 12 in the H-CDR2 region
• amino acid sequence SEQ ID NO: 15 in the L-CDR1 region is the amino acid sequence SEQ ID NO: 15 in the L-CDR1 region
• amino acid sequence SEQ ID NO: 16 in the L-CDR2 region is the amino acid sequence SEQ ID NO: 16 in the L-CDR2 region
• amino acid sequence SEQ ID NO: 17 in the L-CDR3 region is the amino acid sequence SEQ ID NO: 17 in the L-CDR3 region.
• an object of the invention relates to an antibody directed against galectin 9 and inhibitor of the suppressive activity of regulatory T lymphocytes having for CDRs the six CDRS defined by:
• amino acid sequence SEQ ID NO: 12 in the H-CDR2 region is the amino acid sequence SEQ ID NO: 12 in the H-CDR2 region
• amino acid sequence SEQ ID NO: 15 in the L-CDR1 region is the amino acid sequence SEQ ID NO: 15 in the L-CDR1 region
• amino acid sequence SEQ ID NO: 16 in the L-CDR2 region is the amino acid sequence SEQ ID NO: 16 in the L-CDR2 region
• amino acid sequence SEQ ID NO: 17 in the L-CDR3 region is the amino acid sequence SEQ ID NO: 17 in the L-CDR3 region.
• the heavy chain variable region of said antibody has the amino acid sequence SEQ ID NO: 10 and the light chain variable region of said antibody has the amino acid sequence SEQ ID NO: 14.
• an antibody according to the invention can bind specifically to an epitope of galectin 9.
• an antibody according to the invention is capable of bind specifically to the membrane or intracellular galectin 9
• an antibody according to the invention can bind to the epitope recognized by the antibody 2E12 defined above.
• the antibody according to the invention is a chimeric antibody, preferably a murine / human chimeric antibody.
• this murine / human chimeric antibody may comprise the variable domains of the 2E12 antibody as defined above.
• the antibody according to the invention is a humanized antibody.
• the variable domain of this humanized antibody may comprise framework regions of the human acceptor, and possibly human constant domains, and the CDRs of the non-human donor, in particular the CDRs defined above.
• the antibodies of the invention may be produced by any technique known to those skilled in the art, for example, but not limited to, by any chemical, biological, genetic or enzymatic technique taken alone or in combination.
• the specific binding of the antibodies according to the invention directed against galectin 9 can be analyzed according to any known method of the state of the art.
• immunoassays that may be used include western blot techniques, radioimmunoassays, ELISA, sandwich immunoassays, immunoprecipitation assays, precipitin assays, gel diffusion assays. precipitin, immunoradiometric assays, fluorescence immunoassays or complement fixation tests. Such tests are well known to those skilled in the art.
• the inhibitory action of the suppressive activity of regulatory T cells of an antibody according to the invention thus generated can be analyzed according to various techniques known to those skilled in the art. For example, a cell proliferation assay can be performed. It is thus possible to refer to the technique used below concerning the cell proliferation test carried out with the antibody directed against galectin 9, 1G3 or 2E12.
• amino acid sequence of the desired sequence is known, those skilled in the art can readily replicate the antibody by standard polypeptide production techniques.
• such antibodies can be synthesized by a well known solid phase method, preferably using a commercially available peptide synthesizer and following the recommendations of the supplier.
• the antibodies of the invention may be obtained by techniques, well known to those skilled in the art, of recombinant DNA in a suitable expression system.
• expression system means a cellular host and a compatible vector under appropriate conditions, i.e., conditions allowing the expression of the protein encoded by the foreign DNA carried by the vector and introduced into the vector. the host cell.
• the nucleic acid sequence encoding an antibody may be inserted into an appropriate expression vector which will then be introduced into a suitable prokaryotic or eukaryotic host which will produce the desired antibody.
• vector refers to vehicles by which the DNA or RNA sequences encoding the antibody can be introduced into a host cell so as to transform it and to allow the expression (ie, transcription and translation) of the introduced sequence.
• An expression vector is typically a plasmid, a cosmid, an episome, an artificial chromosome, a phage or a viral vector.
• adenoviruses As viral vectors, mention may be made of adenoviruses, retroviruses, herpesvirus and vectors derived from adeno-associated virus (AAV).
• AAV adeno-associated virus
• Such recombinant viruses can be produced by well known techniques, such as transfection of cell lines allowing their encapsidation or transient transfection with plasmids or complementation viruses expressing the necessary missing functions.
• Cell lines for encapsidation are for example PA317, PsiCRIP, GPenv +, 293, etc.
• Detailed protocols for producing such replication-defective recombinant viruses are available in patent applications WO 95/14785, WO 96/22378, US 5,882,877, and the like.
• the host cells are therefore transfected, infected or transformed with a nucleic acid or a suitable vector as described above.
• transformation refers here to the introduction of a foreign gene (extrinsic or extracellular), a DNA or RNA sequence into a host cell such that this host cell expresses the gene or the sequence introduced to produce the desired substance, typically a protein encoded by the introduced gene or sequence.
• Common expression systems include, but are not limited to, host cells and plasmid vectors of E. coli. coli, insect host cells and Baculovirus vectors and mammalian cells and vectors.
• a method of production from a host cell expressing an antibody according to the invention may comprise the steps of: (i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described above into the cell competent host, (ii) culturing in vitro or ex vivo the recombinant host cell thus obtained, (iii) optionally selecting the cells which express and / or secrete said antibody or polypeptide.
• Such host cells can be used for the production of antibodies according to the invention.
• a method of producing an antibody according to the invention may comprise the steps of: (i) culturing the transformed cell described above under conditions appropriate for the expression of the antibody; and (ii) recovering the antibody thus expressed.
• the antibodies can be separated from the culture medium by conventional immunoglobulin purification methods such as, for example, purification. on protein A-Sepharose, by chromato graphy on hydroxylapatite, by gel electrophoresis, by dialysis or by affinity chromatography.
• a human chimeric antibody according to the invention can be produced by obtaining the nucleic sequences encoding the VL and VH domains as mentioned above, by constructing a human chimeric antibody expression vector by inserting the nucleic sequences into an animal cell expression vector having genes encoding the CH and CL domains, and expressing the encoded sequences by introducing the expression vector into the animal cell.
• the CH domain of the human chimeric antibody can be from any region belonging to human immunoglobulin. Preferably, it is the IgG class, and more preferably IgG1.
• the CL domain of the human chimeric antibody can be from any region belonging to human immunoglobulin. Preferably, it is the Kappa class.
• the chimeric or humanized antibodies according to the invention may in particular be obtained by genetic engineering of the antibodies.
• a chimeric antibody can for example be performed by a gene transfection technique or by a recombinant DNA technique.
• a humanized antibody according to the invention can be produced by obtaining the CDR domains as mentioned above, by constructing a human antibody expression vector by inserting the nucleic sequences into an animal cell expression vector having genes encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and expressing the coded sequences by introducing the expression vector into the animal cell.
• the humanized antibody expression vector it may be a type in which a gene encoding an antibody heavy chain and a gene encoding a chain Antibody antibodies exist in separate vectors, either of a type in which both genes exist in the same vector (tandem type). Tandem-type vectors are preferred over the ease of construction of the expression vector, ease of introduction into animal cells etc.
• Tandem-type vectors are preferred over the ease of construction of the expression vector, ease of introduction into animal cells etc.
• pKANTEX93 or pEE18 mention may be made of pKANTEX93 or pEE18.
• the antibodies can be humanized according to various techniques known from the state of the art, for example by CDR grafting, veneering or resurfacing, or by chain shuffling. .
• the recombinant DNA-based technique for the preparation of such antibodies is also known.
• a Fab fragment according to the invention can be obtained by treatment of an antibody, in particular an antibody directed against galectin 9 and inhibitor of the suppressive activity of regulatory T cells, with a protease, papain.
• This Fab fragment may also be produced by insertion of a DNA encoding the Fab fragment of the antibody into a vector usable in a prokaryotic or eukaryotic expression system and the introduction of this vector into the prokaryote or eukaryote appropriate for express the Fab fragment.
• An F (ab ') 2 fragment according to the invention can be obtained by treatment of an antibody, in particular an antibody directed against galectin 9 and an inhibitor of the suppressive activity of regulatory T cells, by a protease, pepsin.
• the F (ab ') 2 fragment can also be obtained by joining together Fab' fragments as described below, by a thioether bond or a disulfide bridge.
• a Fab 'fragment according to the invention can be obtained by treatment of the F (ab') 2 complex of an antibody, in particular an antibody directed against galectin 9 and an inhibitor of the suppressive activity of regulatory T cells, by a reducing agent. , dithiothreitol.
• the Fab 'fragment may also be produced by insertion of a DNA encoding the Fab' fragment of the antibody into a vector usable in a prokaryotic or eukaryotic expression system and the introduction of this vector into the prokaryote or eukaryote suitable for expressing the Fab 'fragment.
• the ScFv fragment according to the invention can be produced by obtaining a cDNA sequence encoding the previously described VH and VL domains followed by the insertion of this DNA into a vector that can be used in a eukaryotic or prokaryotic expression system. and introducing this vector into the eukaryotic or appropriate prokaryote to express the ScFv fragment.
• a humanized ScFv fragment it is possible to use the CDR grafting technique. This technique involves the selection of complementarity regions (CDRs) of a donor ScFv fragment and their graft on the framework of a human ScFv fragment of known three-dimensional structure (see for example WO 98/45322, EP0173494).
• Modifications of the amino acid sequences of the antibodies according to the invention can be carried out. For example, it may be desirable to improve the binding affinity and / or biological properties of the antibody. It is known that when a humanized antibody is produced by simply grafting only VH and VL CDRs of an antibody derived from a non-human animal into the frameworks (FR) of a human antibody, the binding power to the antigen is decreased in comparison with that of an antibody derived from a non-human animal. It is believed that some amino acid residues of the VH and VL of a non-human antibody, not only in CDRs but also in FRs, are directly or indirectly associated with the antigenic binding power.
• “Functional conservative variants” are those in which a given amino acid residue in a protein has been changed without impairing the overall conformation and inhibitory function of the regulatory T cell suppressive activity. Thus, it is possible to replace one amino acid with another having similar properties (eg, polarity, hydrogen bonding potential, etc.) as long as the inhibitory function of the regulatory T-cell suppressive activity is maintained.
• an antibody as defined above directed against galectin 9 and an inhibitor of the suppressive activity of regulatory T lymphocytes, comprising:
• H-CDR1 having 1 or 2 amino acids of difference with the sequence defined by SEQ ID NO: 2,
• H-CDR2 having 1 or 2 amino acids of difference with the sequence defined by SEQ ID NO: 3,
• H-CDR3 having 1 or 2 amino acids of difference with the sequence defined by SEQ ID NO: 4,
• an antibody as defined above, directed against galectin 9 and inhibitor of the suppressive activity of regulatory T lymphocytes has 1, 2 or 3 amino acids of difference with the whole. of the six CDRs of sequences as defined above, that is to say the CDRs of sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 , and SEQ ID NO: 8.
• H-CDR1 having 1 or 2 amino acids of difference with the sequence defined by SEQ ID NO: 11,
• H-CDR3 having 1 or 2 amino acids of difference with the sequence defined by SEQ ID NO: 13,
• an antibody as defined above, directed against galectin 9 and inhibitor of the suppressive activity of regulatory T lymphocytes has 1, 2 or 3 amino acids of difference with the whole. of the six CDRs of sequences as defined above, that is to say the CDRs of sequences SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16 , and SEQ ID NO: 17.
• regulatory T cells can induce inappropriate immunosuppression, which then promotes tumor growth. Numerous studies have thus shown that regulatory T lymphocytes reduce anti-tumor immune responses, in particular by inappropriately inhibiting the activity of effector T lymphocytes, thus favoring the development of cancer-type pathologies.
• galectin 9 is directly expressed by regulatory T lymphocytes while it is only very weakly, even not at all, by effector T cells, the targeting of galectin 9 to specifically inhibit regulatory T cells without the risk of causing depletion of effector T cells.
• the inhibition of galectin 9 by an antibody allows the inhibition of the suppressive activity of regulatory T cells.
• the antibodies according to the invention, directed against galectin 9 and inhibitors of the suppressive activity of regulatory T lymphocytes can therefore be used in the treatment of diseases associated with the suppressive activity of regulatory T cells, in particular the treatment of cancers.
• An object of the invention thus relates to an antibody as described above for its use in the treatment of diseases associated with the suppressive activity of regulatory T cells.
• a particular embodiment of the invention relates to an antibody, as previously described, used in the treatment of cancer.
• cancer treatment is meant any treatment that can, for example, suppress a tumor or metastases, reduce the risk of recurrence, slow tumor development or metastasis, and / or treat the symptoms of the disease.
• the cancers targeted by the present invention are those in which the regulatory T cells exert their suppressive activity.
• the cancers targeted by the present invention are those in which the regulatory T cells are present in a large quantity in the tumor tissue or in the circulation, the expansion of the regulatory T lymphocytes generally being correlated with the increase in their activation.
• the frequency of regulatory T lymphocytes may be evaluated by any method known to those skilled in the art, for example by flow cytometric analysis (FACS) of intra-tumor lymphocytes or circulating lymphocytes or by immunohistological labeling of the tissue. tumor.
• FACS flow cytometric analysis
• an antibody as defined above can therefore be used in the treatment of all types of cancers in which the regulatory T cells exert their suppressive activity.
• Many types of cancers in which the Regulatory T cells exert their suppressive activity have been the subject of studies and are known to those skilled in the art.
• An antibody as defined above can therefore in particular be used in the treatment of cancer, the cancer being chosen from the group consisting of chronic myeloid leukemias, colon cancer, melanoma, uterine cancer, breast cancer, pancreatic cancer. , gastric cancers, ovarian cancer, primary lymphoma of the central nervous system, multiple myeloma, prostate cancer, Hodgkin lymphoma and hepatocellular carcinoma.
• cancers produce significant amounts of exosomes carrying galectin 9 playing an immunosuppressive role, that is to say, inhibiting the immune response and potentially the anti-tumor response.
• cancers producing large amounts of exosomes carrying galectin 9 include virally induced cancers, for example nasopharyngeal carcinomas associated with EBV virus (Epstein-Barr virus), or hepatocellular carcinoma (HCC) associated with HCV (hepatitis C virus) or HBV (hepatitis B virus) (20,21).
• an antibody as defined above can therefore in particular be used in the treatment of cancer, the cancer being a viro-induced cancer, preferably selected from the group consisting of nasopharyngeal carcinomas associated with Epstein-Barr Virus, hepatocellular carcinomas Hepatitis C virus or hepatitis B virus.
• An antibody according to the invention can therefore be used to prevent a recurrence of fibrosis consecutive to hepatitis C.
• the anti-galectin-9 antibody and inhibitor of regulatory T-cell suppressive activity is suitably administered to a patient in need of such treatment.
• the antibodies according to the invention may be used alone or in combination with any other suitable compound.
• An object of the invention relates to a method of treating cancer, associated with the expression of galectin 9 and the suppressive activity of regulatory T cells, comprising administering a therapeutically active amount of an antibody according to the invention to a patient.
• patient is understood to mean a human affected, or caused to be affected, by a disease associated with the suppressive activity of regulatory T cells, including cancer.
• terapéuticaally active amount of an antibody means an amount of antibody sufficient to treat such a cancer, having an acceptable benefit / risk ratio for drug treatment.
• the amount of antibodies and compositions according to the present invention as well as the frequency of administration will be determined by clinical studies, by the physician or by the pharmacist.
• the amount “therapeutically active” specific to each patient may depend on a number of factors such as the nature and severity of the disorder to be treated, the activity of the antibody used, the composition used, the age, the weight , general health status, sex and diet of the patient, mode of administration, duration of treatment (single or multiple doses), combination drugs and other factors well known to medical specialists .
• an antibody directed against galectin 9 and an inhibitor of the regulatory T cell suppressive activity as previously defined is used in combination with a second agent for the treatment of a disease associated with the suppressive activity of regulatory T cells, for example an anticancer agent.
• the antibody when the use is the treatment of cancer, can be used in combination with known therapies against cancer such as, for example, surgery, radiotherapy, chemotherapy or combinations thereof.
• the antibody may be used in combination with adoptive immunotherapy, consisting of one or more effector lymphocyte injections against tumor antigens including EBV antigens.
• other anticancer agents used in combination with the anti-galectin antibody 9 of the invention for cancer therapy include anti-anogenic agents.
• said antibody is co-administered with a cytokine, for example a cytokine that stimulates an anti-tumor immune response.
• An object of the invention therefore relates to a combination product comprising an antibody, as defined above, and an anticancer agent.
• One particular embodiment relates to such a combination product for simultaneous, separate or spread over time use in cancer treatment.
• the antibody is generally formulated as a pharmaceutical composition.
• the pharmaceutical composition comprising an antibody according to the invention may be formulated by methods known from the state of the art, in which the therapeutic molecule is in combination with at least one excipient.
• An object of the invention thus relates to a pharmaceutical composition
• a pharmaceutical composition comprising an antibody as described above and at least one pharmaceutically acceptable carrier.
• a “pharmaceutically acceptable carrier” means any standard pharmaceutical carrier, its administration being tolerable by a patient.
• sterile phosphate buffered saline solutions are pharmaceutically acceptable.
• the pharmaceutically acceptable carriers can usually comprise one or more compounds, for example selected from excipients, preservatives, solubilizers, buffering agents, albumin, etc.
• excipients are, for example, starch, gelatin, stearic acid, calcium or magnesium stearate and the like. Those skilled in the art will be able to determine the compounds suitable for the present composition.
• the form of the pharmaceutical composition, the mode of administration, the dosage and the dosage may, of course, depend on, among other things, the disease to be treated, its symptoms, severity, age, weight and sex. of the patient.
• the pharmaceutical composition according to the invention may be formulated so as to be administered topically, parenterally, nasally, intravenously, subcutaneously / intradermally, conjunctivally, intramuscularly or intraocularly.
• compositions according to the invention may optionally contain pharmaceutically acceptable excipients suitable for them to be injected.
• pharmaceutically acceptable excipients suitable for them to be injected.
• these may be isotonic and sterile saline solutions, monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or a mixture of these salts.
• These compositions may also be dry compositions, in particular dry and frozen, freeze-dried or refrigerated compositions which, after addition, according to the case, of sterile water or physiological saline, constitute injectable solutions.
• the doses used can be adapted according to different parameters, such as, in particular, the mode of administration according to the pathology or alternatively, the duration of the treatment envisaged.
• a sufficient amount of antibody may be dissolved or dispersed in a pharmaceutically acceptable carrier or an aqueous medium.
• Pharmaceutical forms suitable for injection use include sterile water solutions, dispersions, formulations including sesame oil, or aqueous propylene glycol, as well as sterile powders for the extemporaneous preparation of sterile injectable solutions.
• the form used must be sterile and must be sufficiently fluid to be easily injected by means of a syringe. It must be stable under the conditions of production and storage and be protected from contamination by microorganisms, such as bacteria or fungi.
• Solutions of the active compounds may be prepared with water mixed with a surfactant such as hydroxypropylcellulose.
• a surfactant such as hydroxypropylcellulose.
• the dispersions can be made in glycerol, in liquid polyethylene glycols, in a mixture of both or in oils. These preparations generally contain a preservative to prevent the growth of microorganisms under normal conditions of storage and use.
• An antibody according to the invention may be formulated in a composition in neutral form or in salt form.
• Pharmaceutically acceptable salts include acid addition salts, formed with free amino groups of the protein, and formed with inorganic acids such as hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, mandelic, etc. .
• the salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.
• inorganic bases such as sodium, potassium, ammonium, calcium, or iron hydroxides
• organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.
• the solutions may be administered in a manner compatible with the dosage of the formulation and in a therapeutically active amount.
• the drugs can be administered as described above but also in the form of capsules that release them.
• Figure 1 Phenotypic analysis by multiparametric flow cytometry of natural regulatory T cells isolated from human blood.
• Figure 2 Analysis of the suppressive function of natural regulatory T cells isolated ex vivo.
• A Analysis of inhibition of proliferation of PBMCs activated by autologous regulatory T cells in cpm.
• B Cytolytic percentage analysis of PBMCs activated by autologous regulatory T cells.
• Figure 4 Western blot analysis of the expression of the three isoforms of galectin 9 in human regulatory T cells. Hela cells are used as a negative control.
• FIG. 7 Analysis of the inhibition of the suppressive activity of regulatory T lymphocytes by the anti-gal9 1G3 antibody by the analysis of the proliferation of PBMCs in the presence of irradiated C15, in the presence or absence of regulatory T lymphocytes, and in the presence or absence of 1G3 antibodies at a concentration of 1 g / ml.
• FIG. 8 Galectin-induced inhibition of Galectin 9-induced Jurkat apoptosis assay with 1 ⁇ / ml of Galectin-9 S preincubation and 5 ⁇ g / ml antibodies.
• Antibody 9M1 anti-galectin 9
• 9S2-3 anti-galectin 9
• 1G3 anti-galectin 9
• anti-TIM3 antibody anti-TIM3 antibody
• 2E12 anti-galectin 9
• the control corresponds to the culture medium.
• FIG. 10 Analysis of the expression of galectin 9 by flow cytometry (FACS).
• FIG. 11 Relative Proliferation of Convected T-lymphocytes under Different Conditions After 3 Days of Culture (Activated or Unactivated Cells in the Presence of Galectin-9 and with 1G3 ("Anti-X"), a Control Isotype (IgG1), an Inhibitor (Lactose) ) and a control inhibitor (sucrose)).
• Figure 12 Proliferation of PBMC under different conditions after 5 days of culture (cells activated or not, in the presence of 1G3 or a control isotype (IgG1).
• FIG. 13 Viability of PBMCs under different conditions after 5 days of culture (cells activated or not, in the presence of 1G3 or control isotype (IgG1).
• Figure 14 Measurement of the secretion of galectin 9 by conventional CD4 + T ("T conv”) and regulatory T cells ("Tregs”) in non-activated and activated conditions (test performed after 48 hours of culture).
• FIG. 15 Relative proliferation of conventional T lymphocytes in co-culture with autologous regulatory T lymphocytes under different conditions (activated cells, in the presence of 1G3 (“anti-X”), a control isotype (IgG1), an inhibitor (lactose) and / or a control inhibitor (sucrose)).
• Figure 16 Relative proliferation of PBMCs in the presence of regulatory T cells and different concentrations of 1G3 (1 ⁇ / ml, 3 ⁇ / ml, 5 ⁇ / ml).
• Figure 17 Tumor volume under different treatments: PBMCs alone (control); PBMC + Treg + 1G3 (enabled); PBMC + Treg + IgG1 (activated); without PBMC + 1G3 (enabled); without PBMC + IgGl (activated)
• Figure 18 Graphical representation of the weight of mice as a function of days.
• a and B Independent experiments with xenotransplanted SCID mice (50 ⁇ 10 6 PBMCs + 6% Treg) and treated with 1G3 or the IgG1 control isotype (20 ⁇ g / mouse).
• Figure 19 Graphical representation of the weight of mice as a function of days.
• Figure 21 Measurements of tumor volume measured by bioluminescence.
• Figure 24 Comparative cytometric analysis of the expression of different markers after 5 days of conditioning.
• A Expression of CD4 between activated Tconv (light gray), activated with Gal-9 M (medium gray) or Gal-9-S (black).
• B Expression of CD127 between activated Tconv (light gray), activated with Gal-9 M (medium gray) or Gal-9-S (black).
• C Expression of CD25 between Tconv activated (light gray), activated with Gal-9 M (medium gray) or Gal-9-S (black).
• Figure 26 Analysis of the inhibition of the suppressive activity of regulatory T cells by the anti-gal9 1G3 antibody and the anti-gal9 2E12 antibody (at the concentration of 1 g / ml) by analyzing the proliferation of PBMCs co-cultured with regulatory T cells.
• the C15 tumor cell lines are derived from a xenotransplanted EBV-positive CNP (Nasopharyngeal Carcinoma) and continuously propagated in SCID mice subcutaneously every 6-7 weeks. All animal experiments were carried out by qualified personnel, in accordance with the French and European regulations, in the pet shop of the Pasteur Institute of Lille (France). C15 cells were recovered from xenotransplanted tumors and irradiated (5000 rads) before being preincubated with immune cells simulating a tumor context.
• the Jurkat line was established from human T lymphocyte leukemia. It has the phenotype of CD4 + lymphocytes.
• the standard culture medium used is RPMI 1640 (Invitrogen, Paisley, UK) supplemented with 10% human serum AB (BioWest, Nuaillé, France), 2 mM L Glutamine, 1 mM sodium pyruvate, 10 mM acid non-essential amines, 10 mM HEPES, 50 U / mL streptomycin, 50 ⁇ g / mL gentamycin and 50 ⁇ M ⁇ mercaptoethanol.
• the cells were incubated at 37 ° C under a controlled atmosphere (5% CO 2 and 95% moisture) in a Hera Cell 150 incubator (Thermo Electron, Cergy Pontoise, France).
• PBMCs and CD4 + T cells were activated with an anti-CD3 antibody (1 ⁇ g / mL) (Clinisciences, Montrouge, France), which binds to the plate after incubation for 2 hours at 37 ° C. culture, and a soluble anti-CD28 antibody (100 ng / mL) (Clinisciences) added extemporaneously.
• an anti-CD3 antibody (1 ⁇ g / mL) (Clinisciences, Montrouge, France)
• a soluble anti-CD28 antibody 100 ng / mL
• PBMCs Peripheral blood mononuclear cells
• CD4 + T cells were isolated from PBMCs using a negative selection protocol according to the manufacturer's instructions (Miltenyi Biotec, Berlin, Germany). Briefly, the PBMCs are incubated for 10 minutes with a cocktail of biotinylated antibodies directed against CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCR ⁇ / ⁇ and glycophorin A. Anti-biotin magnetic beads are then added during 15 minutes. The cells to be removed are magnetically retained in a Magnetic Activated Cell Sorting column (MACS®) placed in a MACS® separator. The cells to be isolated pass through the column, they are collected and enriched in unmarked cells, depleted of non-targeted cells. Flow cytometry analysis shows that more than 98% of the isolated cells are CD4 + cells. Isolation of regulatory T cells
• CD4 + CD25 + regulatory T cell isolation kit (Miltenyi Biotech, Germany) according to the manufacturer's instructions.
• the CD4 + CD25 + T cell fraction was preserved for flow cytometry and chemoattraction experiments.
• the flow cytometry analysis consistently shows an enrichment greater than 95% of the CD4 + CD25 + fraction.
• Cell proliferation test 1.10 5 cells (PBMCs or CD4 + T cells) were incubated with [methyl 3H] thymidine during the last 18 hours of culture and collected on a fiberglass filter (Printed Filtermat A, Wallac, Turku, Finland) using a collector Tomtec (Wallac). The filter was then sealed in a bag after drying and addition of the scintillation liquid (Beckman Coulter, USA). Proliferation was measured after incubation in the presence of [3H] thymidine (1 ⁇ / ⁇ 8) (PerkinElmer, Courtaboeuf, France) for the last 18 hours before collection. Radioactivity was measured using a ⁇ -counter (1450 Trilux, Wallac, Finland).
• Each proliferation test was performed in triplicate and estimated in "count per minute” (cpm). According to the experiments, the proliferation tests were carried out in the presence of 1 ⁇ g / mL of the short isoform of recombinant Galectin 9 (Gal9S) supplied by Dr. Toshiro Niki (Galpharma, Japan), of 1 ⁇ g / mL or a range of anti-Galectin 9 1G3 antibody, an irrelevant anti-IGgl antibody as a negative control (ebiosciences, UK), 10 ⁇ g / mL C15 exosomes, 5 mM lactose or sucrose (Sigma Aldrich).
• the technique of measurement of cell lysis is based on the use of Cytotoxicity Kit (CytoTox-Glo Assay, Promega, USA) which measures a luciferase activity proportional to cellular proteases released after cytolysis.
• the tests are performed by co-culturing 6.10 5 CD4 + CD25 + and 2.10 5 autologous PBMCs.
• the cells are cultured in 96-well plates to round bottom plates (Maxisorb Nunc, Denmark) in 200 ⁇ ⁇ of culture medium (RPMI-1640, L-Glutamine 2 mM 1%, 0.02 mM sodium pyruvate, 100 U penicillin / mL, 100 ⁇ g / mL streptomycin, 10% AB decomplemented Human Serum) (GIBCO BRL TM, Invitrogen®, GB).
• the cells are activated with 1 ⁇ g / ml of anti-CD3, previously coated on the plates (2 h at 37 ° C.), and 100 ng / ml of anti-CD28.
• reagent aminoluciferin-Glo
• 50 ⁇ ⁇ reagent aminoluciferin-Glo
• the culture plates are incubated for 15 min at room temperature and protected from light.
• a first luminescence measurement is made at the luminometer (Centro LB960, C Berthold Technologies, France), and is proportional to the amount of cells lysed by regulatory T cells.
• 50 ⁇ ⁇ a digitonin solution are deposited into each well to induce complete lysis of cells.
• the plates are then shaken and then incubated for 15 min at room temperature and in the dark. before making the second luminescence measurement.
• the tests are performed in triplicate and the results are expressed as a percentage of lysis.
• Percent Lysis Cell Viability / Total Lysis Average - Background
• Cytolysis Mean lysis induced by Treg lymphocytes - background noise
• Jurkat cells cultured in RPMI 5% fetal calf serum are transferred to a serum-free medium (Hybridoma SFM - Life Technologies) and then incubated in the presence of 30 nM galectin-9 for 24 hours in a 96 well plate well (100,000 cells / well).
• the apoptotic count of cells is done by flow cytometry after labeling with annexin V-APC (allophycocyanin) and propidium iodide.
• the galectin is preincubated for 30 min in the presence of the antibody whose final concentration for the incubation of 24 hours is 10 ⁇ g / ml.
• the exosomes were lysed (10 min on ice) in PY buffer composed of 20 mM Tris HCl, 50 mM NaCl, 5 mM EDTA, 1% Triton X 100, 0.02% sodium azide and a cocktail of inhibitors. proteases (Roche, Basel, Switzerland). After centrifugation (20,000 g, 15 min, + 4 ° C), cell debris was removed and the supernatants collected. Protein concentrations were measured using Bio Rad Protein Assay according to the manufacturer's instructions (BioRad, Marnes la Coquette, France). The exosomes were then analyzed by Western Blot.
• the proteins were separated by SDS PAGE electrophoresis using precolumn gradient gels (12% gradient, Bis Tris, Invitrogen) under standard conditions. The proteins were then transferred to a nitrocellulose membrane (Hybon dTM-C Extra, Amersham Biosciences, UK). The latter was blocked for 1 hour at room temperature in a blocking buffer containing 0.2% AuroraTM blocking reagent (MP Biomedicals, Illkirch Graffenstaden, France), 0.1% Tween20 (Sigma Aldrich) and PBS (IX), and then it was incubated overnight at 4 ° C with a primary antibody to Galectin 9: Galectin-9-CT-L1 1: 100 (supplied by Galpharma, Japan).
• the membrane was washed with blocking buffer and then incubated for 1 hour at room temperature with a peroxidase-conjugated secondary antibody (anti mouse, 1: 10000) (GE Healthcare, Wauwatosa, USA) and washed again with the blocking buffer.
• a peroxidase-conjugated secondary antibody anti mouse, 1: 10000
• Protein-specific signals were visualized using Western Lightning® Plus ECL, a chemiluminescence enhancement kit for the substrate (Perkin Elmer, Boston, MA, USA) and a LAS3000 luminescent image analyzer (Fujifilm).
• Immunophenotyping of cells by flow cytometry was performed using the FACSCalibur flow cytometer. After collection, cells were washed with phosphate buffered saline (PBS) (GIBCO-Life technologies) and labeled with monoclonal antibodies conjugated to fluorochromes (1:10). For each assay, the appropriate control isotypes (monoclonal antibodies) were used for the marker settings. Finally, the data was analyzed with the FlowJo software.
• PBS phosphate buffered saline
• anti-human mouse antibodies were used: CD4-phycoerythrin (PE) -cyanin (Cy) (BD Pharmingen, San Diego, USA), -CD25-PE (Miltenyi Biotech, Germany) and -CD127-FITC (1:20) (Clinisciences, Montrouge, France) according to the manufacturer's instructions.
• RNAs of the regulatory T cells were isolated using the kit "RNeasy minikit II" (Qiagen) according to the manufacturer's instructions. The concentration and purity of the RNAs were measured by the spectrophotometric method (Ultrospec 3000, Pharmacia Biotec). Total RNA was stored at -80 ° C until further use.
• Reverse transcription of mRNA was performed as follows: 2 ug of total RNA were mixed with 5 ⁇ ⁇ of master mix consisting of 1 ⁇ ⁇ oligo dT (Roche Diagnostics, Meylan, France) and 0.1 ⁇ ⁇ RNAsin (40 U / ⁇ , Promega, Charbonippos, France) and incubated at 70 ° C for 5 to 10 minutes.
• ultra-pure distilled water (GIBCO-Life Technologies) was added in order to obtain a final concentration of 10 ng of total DNA ⁇ L.
• the DNA was stored at -20 ° C until further use.
• the transcripts were quantified using real-time PCR (RT-PCR) with the Mx3005PTM sequence detection system (Agilent Technologies, France) in 96-well optical plates (Eurogentec SA, Belgium).
• RT-PCR real-time PCR
• Mx3005PTM sequence detection system Algilent Technologies, France
• Eurogentec SA Eurogentec SA, Belgium
• 10 ⁇ ⁇ of primers specific torque designed for RT-PCR and purchased from MWG-Biotech (Germany), were prepared to a final concentration of 10 pg / ml and then stored at - 20 ° C.
• Household ⁇ -actin, Glyceraldehyde-3-phosphate dehydrogenase (G3PDH), ubiquitin and Hypoxanthine guanine phosphoRibosyl Transferase (HPRT) genes were used as controls in each plate.
• PCR reactions were performed according to the manufacturer's instructions to a final volume of 20 ⁇ ⁇ and a ⁇ .mu.l cDNA (equivalent to 10 ng of total RNA using 2X MES A GREEN qPCR MasterMix Plus for SYBR® 258 Assay (Eurogentech) containing Meteor Taq DNA polymerase, MgC12 (final concentration of 4 mM), dNTPs (including dUTP), SYBR® 260 Green I, stabilizers and passive references required for signal normalization and buffer with optimized components
• the PCR program included initial denaturation and activation of Meteor Taq for 5 minutes at 95 ° C, followed by 40 standard amplification cycles as follows: 15 seconds at 95 ° C (denaturation), 1 minute at 60 ° C ( synthesis and elongation). Fluorescent products were detected at the last step of each cycle. An analysis of the melting curves was carried out immediately after amplification, according to the manufacturer's instructions.
• Quantitative PCR reactions were used to quantify the expression of the galectin 9 gene by regulatory T cells.
• the household genes ⁇ -actin, G3PDH, ubiquitin and Hypoxanthine guanine Phosphoribosyl Transferase (HPRT) were used as controls. All primers were designed for RT-PCR and purchased from MWG-Biotech (Germany).
• a quantitative analysis was performed based on the "cycle threshold" (CT) value or cycle threshold for each well and calculated using the MxPro software. Each individual value was normalized using the average of the 4 household genes
• a recombinant protein representing the C-terminal portion of human galectin 9 was used as an immunogen (residues 191-355 of the long isoform of galectin-9). It was produced in E. coli as a GST fusion protein. After separation of the GST tag, the protein was purified by exclusion chromatography.
• mice Five female BALB-c mice, eight weeks old, were immunized with the C-terminal portion of galectin 9 mentioned above.
• 40 micrograms of protein were injected intraperitoneally at day 0, 22, 37 and 54 in combination with Freund's complete adjuvant for the first injection, or with incomplete Freund's adjuvant for injections. following.
• the quality of the immunization was evaluated by ELISA, described below, on serum samples from the immunized mice.
• the same recombinant C-terminal galectin preparation was used for the immunizations on the one hand and for the ELISA tests on the other hand. These tests showed good immunization in the five treated mice.
• mice that gave the best response were sacrificed and their splenocytes were collected. These splenocytes were used for fusion with Sp2 / 0 murine myeloma cells, either in liquid medium or in semi-solid medium, with respective ratios of 5: 1 and 2: 1. Hybridoma supernatants were then were evaluated by an ELISA test, performed as the previous one and as described below, on the recombinant galectin 9 preparation mentioned above.
• the ELISA test was performed as follows.
• the recombinant protein representing the C-terminal portion of galectin 9 was adsorbed into wells of 96-well microtiter plates (50 ng / well) (Greiner Bio-One, Courtaboeuf, France) as follows: solution in 0.05 M carbonate / bicarbonate buffer at pH 9.6 and incubation in the wells for 1h at room temperature. After washing with PBS containing 0.1% Tween-20, the wells were saturated with 3% bovine serum albumin (BSA) in solution in PBS at room temperature for 1h. They were then incubated with mouse sera or hybridoma supernatants to be tested.
• BSA bovine serum albumin
• Sera and hybridoma supernatants were diluted in PBS with 1% BSA and then incubated in wells at room temperature for 2h. After a washing step with 0.1% PBS-Tween-20, the plates were treated with a peroxidase-labeled secondary antibody (goat anti-mouse). The final revelation takes place after addition of substrate (3,3 ', 5,5'Tetramethylbenzidine or TMB, Thermo Fisher Scientific) and absorbance measurement at 405 and 620 nm on a Multiskan Ex microplate reader (Thermo Fisher Scientific) .
• substrate 3,3 ', 5,5'Tetramethylbenzidine or TMB, Thermo Fisher Scientific
• absorbance measurement at 405 and 620 nm on a Multiskan Ex microplate reader Thermo Fisher Scientific
• Immunodeficient mice are first splenectomized and then xenotransplanted with a CNP C666-1 cell line modified to express luciferase, which makes it possible to follow tumor growth in bioluminescence by imaging on a vigilant animal.
• the immune system of the mice is reconstituted and humanized following the injection of human PBMCs more or less enriched in regulatory T cells (2% of regulatory T lymphocytes in the PBMCs at the origin, and addition of 6 to 10% of T cells regulators in PBMCs).
• mice SCID mice aged 6 to 8 weeks underwent total splenectomy. After 7 days, these same mice are xenotransplanted subcutaneously with cells of a C666-luc tumor line (expressing luciferase) derived from a Nasopharyngeal Carcinoma (NPC). On the same day, the mice receive 30 to 50 million intraperitoneal PBMCs for the reconstitution of the immune system, enriched or not with 10% of regulatory T lymphocytes and, depending on the animals, 2, 20 or 200 ⁇ g of IgG1 antibody or of 1G3 antibodies subcutaneously according to the following scheme (3 mice / group):
• Gpl Isotype IgG1 (not reconstituted)
• Gp2 1G3 (not reconstituted)
• Gp3 reconstituted + 10% Treg + IgGl isotype
• mice receive 3 ⁇ g of CPG-ODN2216 (marketed by MILTENYI BIOTECH) in order to activate the immune response.
• mice are boosted with CPG-ODN2216 (marketed by MILTENYI BIOTECH) and, depending on the groups presented above, a booster of 1G3 or IgG1 antibodies.
• a blood sample is taken to verify the reconstitution by flow cytometry (see protocol page 58). After 28 days of measurement, the mice are sacrificed, the tumors recovered and cryopreserved in preparation for the preparation of Immuno-Histochemistry or Immunofluorescence slides and a blood sample is again taken for analysis by flow cytometry.
• mice are regularly weighed [from day 5 to day 28].
• the volume of the tumor was also measured by analysis of the bioluminescence emitted by the tumor cells that express the luciferase gene, after injection of luciferin. Luminescence is measured via the use of the mouse bioluminescence measurement system (IVIS LUMINA).
• IVIS LUMINA mouse bioluminescence measurement system
• Tumors of 1G3-treated mice or the IgG1 control isotype were weighed after sacrifice. The mass is expressed in grams.
• galectin 9 is capable of inducing the differentiation of naive CD4 T cells into T regs (Seki, Oomizu et al., 2008), which reinforces the importance of this lectin in exhaustion phenomena. the anti-tumor immune response.
• T-cells conv were cultured for 3 or 5 days under activation or non-activation conditions, with or without an isoform of galectin 9 (Gal-9 S or Gal-9 M), with or without 1G3. After these conditioning phases, the cells are recovered, washed and analyzed by flow cytometry and co-cultured with PBMCs or autologous converts. The culture medium was also recovered for ELISA analysis.
• the protocol is as follows: PBMCs were isolated by Ficoll gradient purification and conventional T cells were isolated on magnetic columns according to the supplier's protocol (Miltenyii Biotech, T Cell Isolation kit).
• PBMCs Part of the PBMCs are stored under non-activation conditions and the autologous Tconv are cultured for 5 days under activation or non-activation conditions.
• Activation is by anti CD3 ( ⁇ g / mL) (provided by the team of Anne Tsicopoulos, CIIL) previously coated on the plates (2H at 37 ° C) and anti CD28 (100ng / mL) ) (Clinisciences, France).
• T cells are conditioned or not by the isoforms of Gal-9 S or M at 2 ⁇ g / ml in the presence or absence of the 1G3 antibody at 3 ⁇ g / ml. After 5 days, conditioned Tconv are washed, viability is established by trypan blue counting * (see below), the supernatant is recovered for ELISA assays and 10 5 cells are analyzed by flow cytometry to to measure the expression of markers CD4, CD25 and CD127. The rest of the conventional cells are brought into contact with the autologous PBMCs that had been stored for the MLR test.
• MLR mixed lymphocyte reaction
• PBMCs autologous PBMCs (1: 1 ratio)
• the cells are activated with ⁇ g / ml anti CD3, previously coated on the plates (2H at 37 ° C) and anti CD28 (100 ng / mL).
• the vital azo dye (Trypan Blue) to stain dead cells.
• the count is evaluated by incorporating 1 volume of 0.4% trypan blue solution (Sigma, USA) for a cell suspension volume. After 3min, the mixture is deposited on a blade of Thomas. The dead cells that appear in blue and the living, refractive cells are counted. The measurements are made in triplicate and the results expressed as percentage of living cells. Measurement of the secretion of Galectin 9 by ELISA
• Tregs 100 ⁇ / ⁇ 3 ⁇ 4 culture supernatant activated or non-activated Tregs are deposited in duplicate, and incubated for 2 hours at room temperature. Tregs are classically activated by anti CD3 ( ⁇ g / mL) (provided by the team of Anne Tsicopoulos, CIIL) previously coated on the plates (2H at 37 ° C) and the anti CD28 (100ng / mL ) (Clinisciences, France).
• anti CD3 ⁇ g / mL
• CIIL Anne Tsicopoulos
• the plates are then read spectrophotometer at a wavelength of 492 nm (Multiskan Ex, ThermoLabsystems, France).
• PBMCs 2.10 5 of autologous PBMCs are deposited in co-culture.
• the cells are activated [activation by anti CD3 ( ⁇ g / mL) (provided by the team of Anne Tsicopoulos, CIIL) previously coated on the plates (2H at 37 ° C) and anti CD28 (100ng / mL) (Clinisciences, France)] or not according to the conditions and they are incubated with the 1G3 antibody or the 1G1 control isotype at the concentration of 3, 6 or 12 ⁇ g / ml.
• CD3 ⁇ g / mL
• CIIL anti CD28
• the culture is in flat-bottomed 96-well plates with opaque "walls" (Corning3610, Corning Incorporated, USA) in ⁇ of culture medium (DMEM + 4.5g / L glucose + L-glutamine, 100U / mM penicillin, 10 (g / mL streptomycin, 10% human AB decomplemented serum) (GibcoBRLTM, life technologies, GB).
• DMEM + 4.5g / L glucose + L-glutamine, 100U / mM penicillin, 10 (g / mL streptomycin, 10% human AB decomplemented serum) GibcoBRLTM, life technologies, GB.
• the assay uses the Promega CellTiter-Glo Lumenscent Cell Viability Assay Kit (Promega coorporation, USA), which uses luciferase activity, in the presence of oxygen, to measure cellular metabolism (ATP) as an indicator of cell viability.
• ATP cellular metabolism
• Anti-human monoclonal primary antibodies coupled to fluorochromes anti-CD4-PE (Phycoerythrin) -C (Cyanine) 5 (BD PharmingenTM, USA), anti-CD25-PE (Miltenyi Biotec, France), anti-CD 127-FITC (Fluorescein IsoThioCyanate) (Clinisciences, France).
• the cells (2.10 5 ) are taken up in a volume of ⁇ of sterile PBS (GIBCO BRLTM, Invitrogen®, GB) and incubated for 30 min at room temperature and in the dark with ⁇ , anti-CD4-PC, ⁇ , anti-CD25-PE and 4 ⁇ , anti-CD127-FITC.
• the labeled cells are then taken up with 400 .mu.l of PBS and the fluorescence is analyzed by Facscalibur flow cytometry (FACS Flow Supply System, Becton Dickinson, USA).
• the cytometry results are analyzed by the Flow Jo software (Tree Star Incorporation, USA).
• the cells are fixed by adding ⁇ of 4% PFA (Sigma Aldrich, USA) for 10 min and then they are taken up in 200 ⁇ l of PBS before the cytometer analysis.
• the proliferation assays were performed on 10 May PBMCs cultured for 48 hours.
• the cells are cultured in round bottom 96-well plates (Nunc, Denmark) in 200 ⁇ l of culture medium (RPMI-1640, 2 mM L-glutamine 1%, 0.02 mM sodium pyruvate, 100 ⁇ M / m penicillin, 10 ⁇ g).
• the deletion tests are carried out by MLR (mixed lymphocyte reaction) by co-culturing 6.10 4 Tregs with 10 5 autologous PBMCs, in the presence or absence of the 1G3 or 2E12 antibody.
• the cells are activated with ⁇ g / ml anti CD3, previously coated on the plates (2H at 37 ° C) and anti CD28 (100 ng / mL).
• the proliferation is evaluated by incorporation of luCi / well of tritiated thymidine (H Th) (GE HEALTHCARE, France), 18H before the end of the culture. After 48 hours, the plates are filtered on glass fiber filters (Perkin Elmer, France). The filter is incubated in a liquid scintillation liquid (Beckman Instruments Inc., Ready safe, USA) and read on the scintillation counter (1450 Trilux, Wallac, Finland). The results are finally expressed in counts per minute (cpm).
• Flow cytometry analysis (FACS) of the PBMCs shows that the CD4 + CD25 + CD127 regulatory T cells represent 1% of the total PBMCs (results not shown).
• FACS analysis of phenotypic markers of autologous regulatory T lymphocytes isolated ex vivo indicates that 95% of regulatory T cells are CD4 + CD25 + and that of these cells, 90% are CD 127- or CD1271ow and more than 86% FoxP3 + ( Figure 1).
• Activated regulatory T cells have suppressive activity
• the suppressive activity of human regulatory T lymphocytes isolated ex vivo from the blood of healthy donors was characterized by two complementary functional assays: a proliferation suppression test of autologous regulatory T cell activated PBMCs and a test of cytolysis of PBMCs activated by autologous regulatory T cells (Figure 2).
• Figure 2A shows that single activated PBMCs proliferate well in vitro while ex vivo isolated regulatory T cells are anergic, even after activation.
• the proliferation of activated PBMCs decreases by more than 24% in the presence of activated autologous regulatory T lymphocytes at a ratio of 4: 2 (see Fig. 2A).
• the proliferation test (MLR) thus clearly demonstrates that the regulatory T lymphocytes isolated ex vivo and under activation conditions have immunosuppressive activity.
• galectin 9 by effector T cells decreases upon activation; the expression of galectin 9 by regulatory T cells increases upon activation
• FIG 14 shows that whereas conv T cells and non-activated regulatory T cells produce very small amounts of galectin 9 ( ⁇ 10 ⁇ g / ml: below the detection threshold of an ELISA test) the lymphocytes Activated human regulators are able to synthesize and secrete galectin 9 into the extracellular medium. By elsewhere, this secretion of galectin 9 is significantly greater when regulatory T cells are activated, linking this secretion to their suppressive function.
• Figure 5 demonstrates that the ratio of conventional CD4 + T cells to regulatory T cells decreases considerably during activation.
• constitutively activated effector T cells having an antitumor action, will not be the target of the anti-gal9 antibody.
• the activated and therefore functional regulatory T lymphocytes will be a preferred target of the anti-gal9 antibody.
• C15 tumor cells indeed leads to a stop of their proliferation.
• C15 cells are anergic.
• C15 tumor cells indeed induces a decrease in the cellular proliferation of human PBMCs. It is then demonstrated that the presence of regulatory T cells in the co-culture of PBMCs and C15 induces a further significant decrease in proliferation by about 56%.
• FIG. 7 clearly shows that, unexpectedly, the presence of the 1G3 antibody makes it possible to restore the proliferation of PBMCs. It is thus suggested that the 1G3 antibody neutralizes galectin 9 present on, and expressed by, regulatory T cells. Therefore, it is emphasized that the 1G3 antibody inhibits the suppressive activity of regulatory T cells.
• Anti-galectin 9 1G3 antibody has superior efficacy to other anti-galectin antibodies 9 To compare the effect of the 1G3 antibody with other anti-galectin 9 antibodies, or even an anti-Tim 3 antibody, on the inhibition of regulatory T cell suppressive activity, several tests were performed.
• FIG. 8 presents the results of the test on the effect of the antibodies tested on Jurkat apoptosis induced by recombinant galectin-9.
• the antibodies tested correspond to the antibody 9M1 (anti-galectin 9), 9S2-3 (anti-galectin 9), 1G3 (anti-galectin 9), an anti-TIM3 antibody, 2E12 (anti-galectin 9).
• the protection against apoptosis of Jurkat is better with the 1G3 antibody than with the other anti-galectin 9 9S2-3 and 9M1 antibodies or the anti-TIM3 antibody.
• FIG. 9 presents the results of the test on the effect of the antibodies tested on the proliferation of human PBMCs previously treated with galectin 9.
• the antibodies tested correspond to the antibody ECA-42 (anti-galectin 9), 1G3 ( anti-galectin 9), 2E2 (anti-TIM3) and 2E12 (anti-galectin 9).
• ECA-42 anti-galectin 9
• 1G3 anti-galectin 9
• 2E2 anti-TIM3
• 2E12 anti-galectin 9
• Figure 11 shows the results of the test on the effect of galectin 9 and 1G3 on conv T-cells (CD4 + T) by measuring proliferation in freshly isolated cells, via the incorporation of tritiated thymidine during the last 18 hours. of culture. The results are also given in counts per minute (CPM).
• 1G3 antibody or its control isotype was analyzed on PBMCs by measuring the proliferation of freshly isolated cells extracted from the blood of 3 donors by incorporation of tritiated thymidine during the last 18 hours of the culture. The results were obtained in CPM. It is observed in FIG. 12 that PBMCs isolated ex-vivo proliferate in vitro under activation conditions. It should also be noted that the 1G3 antibody and its IgG1 control isotype do not affect the proliferation of PBMCs, even with large doses of monoclonal antibody (from 3 to 12 ⁇ g / ml). Analysis of the cytotoxic effect of the 1G3 antibody and its control isotype on the viability of PBMCs
• the dose increase of mAb 1G3 and its control isotype (1G1) were analyzed by measuring the viability of freshly isolated PBMCs from 3 donors per luminometric analysis. Mitochondrial metabolism was measured during 5 days of activation. The results were obtained in RLU.
• Figure 13 shows that PBMCs isolated ex vivo maintain and increase their viability in vitro under activation conditions. This also demonstrates that 1G3 and 1G1 isotype do not alter the viability of PBMCs, even at high antibody doses (from 3 to 12 ⁇ 8 / ⁇ ).
• the 1G3 antibody does not affect the proliferation or viability of PBMCs and human CD4 + Ts.
• the risk of a side effect such as the induction of an immunosuppression that would be favorable to tumor progression, is reduced.
• This allows a better maintenance of immune defenses, including anti-tumor, in the patient treated according to the invention.
• the potential for inhibition of regulatory T cell function by the 1G3 antibody was analyzed by proliferation assays of a mixed leukocyte reaction of conventional T and regulatory T cells.
• photographs of tumors also highlight the positive effect of 1G3 which induces a limitation of tumor growth compared to its control isotype.
• Galectin 9 has been shown to induce differentiation of naive T4-like CD4 T cells (Seki et al, Galectin-9 suppresses the generation of Thl7, promotes induction of regulatory T cells, and regulates experimental autoimmune arthritis; Immunol 2008 Apr; 127 (l): 78-88, doi: 10.1016 / j.clim.2008.01.006, Epub 2008 Feb 20), which reinforces the importance of this lectin in the phenomena of exhaustion of the response. anti-tumor immune.
• Figure 24 therefore indicates that preincubation of Tconv with the M or S form of Galectin 9 increases the expression of CD4 and CD25 markers in favor of a phenotype. possibly more suppressive. Nevertheless, this hypothesis is only admissible if it is correlated with a suppressive activity of these conditioned Tconv, allowing to validate the suppressive phenotype, which is the case (cf figure 25). To conclude, it is demonstrated that Galectin 9 induces a conversion of conventional CD4 + T cells to immunosuppressive CD4 + T cells. The 1G3 antibody is capable of neutralizing the induction of this conversion and thus promoting the maintenance of an anti-tumor immune response in the patient treated according to the invention. Analysis of the neutralizing effect of 2E12 on the suppressive activity of Tregs
• the potential for inhibition of the regulatory T cell suppressive function by the 2E12 antibody was analyzed by proliferation assays of a mixed leukocyte reaction of regulatory T cells with autologous PBMCs (2 independent donors).
• the results, visible in FIG. 26, demonstrate that a 2-hour preculture of regulatory T cells with 2E12 was sufficient to reverse the inhibitory effect of cell proliferation by regulatory T cells.
• the antibody 2E12 just like 1G3, is capable of reversing this inhibition.
• the efficacy of 2E12 is significantly lower than 1G3 however, in that a slight decrease in PBMC proliferation is observed, in contrast to 1G3.
• CD4 (+) CD25 (+) immunoregulatory T cells gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity. 2002 Feb; 16 (2): 311-23.

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